This revised application is being submitted in response to the program announcement (PA) "Exploratory/ Developmental (R21) Bioengineering Research Grants (EBRG PA-03-058). Cardiovascular disease is the number one cause of death in the United States. Adult cardiomyocytes do not proliferate and therefore cardiac muscle cannot regenerate when the cells are permanently injured. We have recently isolated several pure populations of cardiomyocytes from murine ES cells which express cardiac specific genes. We propose to use these cells as a renewable source in tissue engineered constructs. To create these constructs, the cells will be mixed with collagen and fibronectin. The constructs will be exposed to multiple mechanical or electrical stimulation regiments using a custom designed device. [unreadable] [unreadable] As the heart develops, gene expression of ventricular cardiomyocytes changes to that of terminally differentiated cardiomyocytes. We are hypothesizing that mechanical load and electrical stimulation will induce our genetically selected cardiomyocytes to become better organized and more highly differentiated. Preliminary data has provided proof of concept for the creation of a construct using either HL-1 cardiomyocytes or the genetically selected ES cells. This data also shows that a mechanical load can be applied to the constructs using the device and that the mechanical load has a direct effect on the morphology of the construct and its cellular components. The proposed research defines a plan that will enable us to optimize the conditions for creating a cardiac tissue engineered construct and to investigate the effect that mechanical load and electrical stimulation has on the differentiation of ES cardiomyocytes. Reverse transcription PCR will be utilized to evaluate the effect that mechanical and electrical stimulation has on cardiac gene expression. The cellular morphology of the construct will be assessed with histology, immunohistochemistry, and transmission electron microscopy. In addition, the mechanical properties of the construct will be tested and compared to normal heart muscle. The significance of this work is that if we are successful in our goals then this methodology could be applied to the use of human ES cells for the therapeutic repair of injured cardiac muscle or the creation of artificial cardiac tissue for surgical repair or as a source of tissue for reconstructive surgery of congenital birth defects. [unreadable] [unreadable] [unreadable]